EPA/CSPA Technical Meeting on the OECD 

Method and QACs

August 10, 2011 at the EPA ESC 

1

AM: 8-12 

• Introductions, scope, agenda – 10 min

• Project Leads and Points of Contact

• Meeting Minutes

• Impact of potential changes to the OECD 

method on the approval process, the 

collaborative study, and implementation – 20 min 

• Highlights of the QAC/OECD Method Data to 

Date (1 hr)

• Industry data

• EPA Data

• Problem Definition 

• Review of the Research Priority List (30 min)

• Break – 15 min

• Lab demonstrations and discussion (1.5 hr)

• Carrier Screening

• Hard Water Preparation

• Test Microbe Harvest

• Neutralizer Confirmation Method

• Alternative Procedures (modifications) 

• Upcoming OECD Test Guidelines Meeting – discuss during lunch 

PM: 1-5

• Down-select Research Projects – 1.5 hr

• List and Prioritize (the top 3-4)

• Test Microbes

• Order

• SOPs and Modifications

• Research Study Protocols

• Break – 15 min 

• Aspects of the Test Designs – 1 hr

• Product Treatments

• Target Performance Standard

• No. of Replications

• Controls

• Number of Laboratories

• Data Analysis

• Responsibilities – 15 min 

• Organizations/Laboratories

• Study Matrix by Project

• Timeline/reporting – 15 min 

• Additional Update Meetings – 15 min 
2

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This information is distributed solely for the 

purpose of pre-dissemination peer review 

under applicable information quality 

guidelines. It has not been formally 

disseminated by EPA. It does not represent 

and should not be construed to represent 

any Agency Determination or Policy.

The information provided should not be 

cited or referenced.

For Official Use Only/Do not Cite or Distribute
OECD Quantitative Method

Background

3

• The OECD Quantitative Method for Evaluating Bactericidal 

Activity of Microbicides (OECD method) recently underwent 

an international ring trial.

• EPA is interested in adopting the OECD methods for 

regulatory purposes and is pursuing implementation. 

• Based on the OECD guidelines, MLB developed and posted 

SOP MB-25 (OECD Quantitative Method for Evaluating 

Bactericidal Activity of Microbicides).

• MLB is conducting demonstration studies.

• Collaborative study

• Implementation Workshop
4

OECD Quantitative Method

Key Features

• Quantitative and “closed” to avoid wash-off of viable 

microorganisms.

• Carriers - brushed stainless steel disks. 

• Default diluent – 375 ppm hard water.

• Contact times that reflect the realities of field 

applications.

• No more than 5 minutes for vegetative bacteria, 

mycobacterium, viruses, and fungi.

• 20 2oC as default temperature.

• Includes a standard soil load.

• 5% mixture of high molecular weight protein, low 

molecular weight protein and mucin

• Uses surrogates for all classes of microorganisms.
OECD Quantitative Method

Key Features

1 cm brushed 

stainless steel disk

10 μL dried 

inoculum

50 μL 

disinfectant

Vial with 

inoculated carrier

5

Count colonies and 

determine log density 

for control and treated 

carriers.
OECD Quantitative Method

Demonstration Study

•Objectives: 

1. Gain working experience.

2. Evaluate the method in-house.

3. Generate quantitative efficacy data for 

EPA-registered products.

4. Assess the technical quality and clarity of 

MLB SOP MB-25.

6
OECD Quantitative Method

Demonstration Study

• Evaluated 13 registered liquid antimicrobial products

• Represent five common classes of active ingredients

• 12 hospital disinfectants,1 sanitizer

• Evaluated products against Staphylococcus aureus and 

Pseudomonas aeruginosa

• OECD parameters (5 min., 375 ppm hard water, 3-part organic 

soil load) 

• 1 replication per product

• 3 treated carriers, 4 control carriers

• The control target range was 5.5 to 6.5 logs per carrier; 

assumes a 5 log performance standard.

7
OECD Quantitative Method

Demonstration Study

• To calculate LR values:

• Log density (LD) of the control carriers minus LD treated 

carriers 

• To provide a LR estimate in cases where the dilutions (for 

treated carriers) were too numerous to count (e.g., lawns)

• 1,000 was substituted at the least concentrated dilution

• The LR was then estimated as a value ≤ LD of control carriers –

log10(1,000)

• In treated carrier sets with complete kill, the LR is listed as ≥ 

average carrier count LD.

8
OECD Quantitative Method

Demonstration Study

• Staphylococcus aureus

• Controls: 6.7 0.1

• Carrier counts were slightly above the prescribed 

upper limit in the OECD method.

• In future testing, dilution of the S. aureus culture 

(e.g., 1:1, 1:5) prior to carrier inoculation will be 

necessary.

• Pseudomonas aeruginosa

• Controls: 6.1 0.2

9
* *

†

* * * * *

† †

†

* * * *

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

A B C D E F G H I J K L M

Log10 Reduction

Log Reduction Values for S. aureus using Label Claim

and OECD Test Parameters

Label Claim

OECD

Hydrogen 

peroxide

Phenol Sodium 

hypochlorite

Quaternary ammonium 

compounds

Quat + 

HCl

Citric 

acid

Test Chemicals

* = Value listed represents complete kill. 

† = Value listed represents a value of 1,000 that was substituted at the least concentrated dilution because growth on the filter was a lawn.
* * *

† † † †

* * * *

* *

† † † †

†

* * * *

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

A B C D E F G H I J K L M

Log10 Reduction

P. aeruginosa Log Reduction using Label Claim

and OECD Test Parameters

Label Claim

OECD

Hydrogen 

peroxide

Phenol Sodium 

hypochlorite

Quaternary ammonium 

compounds

Quat + 

HCl

Citric 

acid

Test Chemicals

* = Value listed represents complete kill. 

† = Value listed represents a value of 1,000 that was substituted at the least concentrated dilution because growth on the filter was a lawn.
OECD Quantitative Method

Conclusions

• Control carrier counts were extremely 

consistent for both test organisms.

• Data (recovered bacteria) for treated 

carriers were consistent within a 

product.

• No data from treated or control carriers 

considered to be an outlier.

12
OECD Quantitative Method

Conclusions

• Based on this study, method performance was 

deemed acceptable.

• Data indicate a range of LR values, depending 

on product.

• LR values for quaternary ammonium-based 

products were consistently low (< 5).

• Minor changes to the SOP are necessary 

including the need for preliminary testing.

13
OECD Quantitative Method

Next Steps

• To further verify method performance, 

MLB recommends a multi-lab 

evaluation/validation-style study.

• OECD method currently being used to test 

Mycobacterium terrae against 

tuberculocidal chemicals.

• Final decisions on performance standards 

are pending.

14
OECD Quantitative Method

Collaborative Study Design

• Use MLB SOP MB-25

• 8-10 labs with previous training

• 6 products, across actives

• OECD default parameters

• 4 or 5 log performance standard

• Two microbes

• Test three products per day

• Three reps

• Kick-off in June 15
16

Organism Replicate Test Day Test Chemical

S. aureus

1 1 D, C, F

2 E, B, A

2 3 B, D, E

4 F, A, C

3 5 B, F, D

6 C, A, E

P. 

aeruginosa

1 7 C, B, D

8 F, E, A

2 9 F, B, D

10 A, E, C

3 11 F, E, C

12 A, D, B

OECD Quantitative Method

Collaborative Study Design
OECD Quantitative Method

Technical Discussion and Laboratory Workshop

• Proposed for Summer 2011 

• Provide updates on the UDM and conduct associated 

laboratory demonstrations

• Minimum and maximum carrier counts

• Performance standards

• Further standardization

• Phase-out of the UDM

• OECD Method Implementation

• Laboratory demonstrations

• Regulatory status

• Science Advisory Panel

• Timeline for implementation 17
OECD Quantitative Method

18

Questions/Comments?
Treatment Brushed Smooth Total Processed

Controls 4 4

QAC-1 3 3 20

Bleach 3 3

Treatment Brushed Smooth Total Processed

Controls 4 4

QAC-2 3 3 20

Bleach 3 3

Treatment Brushed Smooth Total Processed

Controls 4 4

QAC-3 3 3 20

Bleach 3 3

Basic design for brushed versus 

smooth carriers
Treatment Medium 1 Dil. TSB Total Processed

Controls 4 4

QAC-1 3 3 20

Bleach 3 3

Treatment Medium 1 Dil. TSB Total Processed

Controls 4 4

QAC-2 3 3 20

Bleach 3 3

Treatment Medium 1 Dil. TSB Total Processed

Controls 4 4

QAC-3 3 3 20

Bleach 3 3

Basic design for Medium 1 & 2 vs. 

Diluted TSB (P. aeruginosa)
Treatment 1 mL with brushed Total Processed

Controls 4

14

QAC-1 3

QAC-2 3

QAC-3 3

Bleach 3

Treatment 1 mL with smooth Total Processed

Controls 4

14

QAC-1 3

QAC-2 3

QAC-3 3

Bleach 3

Basic design for Medium 1 vs. 

Diluted TSB
Treatment Default Conditions 10 min/DI/no OSL Total Processed

Controls 4 4

QAC-1 3 3 20

Bleach 3 3

Treatment Default Conditions 10 min/DI/no OSL Total Processed

Controls 4 4

QAC-2 3 3 20

Bleach 3 3

Treatment Default Conditions 10 min/DI/no OSL Total Processed

Controls 4 4

QAC-3 3 3 20

Bleach 3 3

Basic design for two test parameter 

sets